Human cytochrome P450 aromatase (P450arom), an integral membrane hemeprotein of the endoplasmic reticulum, catalyzes the synthesis of estrogens from androgens in the presence of P450-reductase. Despite intense biochemical and biophysical investigations for the past 35 years, the structure-function relationships of P450arom and its catalytic mechanism remain poorly understood. Inhibition of estrogen biosynthesis by P450arom inhibitors is an effective therapy for hormone-dependent breast cancers. Attempts to obtain diffraction-quality crystals of human P450arom have so far been unsuccessful. We have grown single crystals of the androstenedione-complex of the full-length, highly active P450arom purified from human term placenta, gathered complete diffraction data to 2.90E resolution and obtained a solution for the structure. Here, we propose to launch an investigation in order to determine of the structure-function relationships of human P450arom. Our hypothesis is that analysis of the atomic structures of human P450arom-ligand complexes in terms of their functional properties will lead to the elucidation of the origin of substrate and inhibitor specificities, roles of the catalytically important residues, nature of the reaction intermediates, as well as the mechanism of action, and that ligand design and optimization guided by these structural basis will lead to novel high-affinity inhibitors that are exclusive for the target. The specific aims to test the hypothesis are to determine the crystal structures of the complexes of P450arom with its (1) natural substrates androstenedione, testosterone, and 161-hydroxy-testosterone, as well as with (2) the inhibitors exemestane, letrozole, formestane, anastrozole, fadrozole and aminoglutethimide. These findings will reveal the molecular mechanism for substrate and inhibitor specificities and help build a structure-activity database based on the atomic level descriptions of the enzyme-ligand interactions. Next, in specific aim 3 we will investigate the enzyme mechanism by combining the data from aims 1 and 2 with structural data on reaction intermediates. By initiating the aromatization reaction in an enzyme-substrate complex crystal with X-ray photoelectrons during diffraction and in situ monitoring of the Soret band transition with a spectrophotometer, we plan to capture structural snap shots of the reaction intermediates. Completion of these goals will lead to the implementation of specific aim 4 - discovery of new inhibitors through docking, design, synthesis, testing, and optimization, in collaboration with chemists. As a future direction of the project, we plan to crystallize a complex of P450arom with P450- reductase for investigating the mechanism of redox reactions by electron transfer. PUBLIC HEALTH RELEVANCE: Aromatase is a unique enzyme that makes all estrogens in the human body. We propose a research plan to unravel the molecular details of how aromatase works and how aromatase inhibitors prevent it from making estrogens. Results from this investigation will form the basis for future discovery of novel breast cancer drugs that are highly specific for aromatase but cause minimal side effects.